Cell wall proteins play important roles in regulating cell wall extensibility which in turn controls cell enlargement. Among cell wall proteins studied to date, expansins are unique in their ability to induce immediate cell wall extension in vitro and cell expansion in vivo. Expansins are extracellular proteins that promote plant cell wall enlargement, evidently by disrupting noncovalent bonding between cellulose microfibrils and matrix polymers (McQueen-Mason, S., et al., (1994) Proc. Natl. Acad. Sci USA 91:6574-6578; McQueen-Mason, S., et al., (1992) Plant Cell 4:1425-1433).
Since their first isolation from cucumber hypocotyls, expansin proteins have been identified in many plant species and organs on the basis of activity assays and immunoblotting. Examples include tomato leaves, oat coleoptiles, maize roots, rice internodes, tobacco cell cultures, and various fruits. The original sequencing of cucumber expansin cDNAs has impacted our understanding of expansins in several respects. First, expansin genes have now been identified in many other plant species, and they appear to be restricted largely to the plant kingdom. Second, expansins comprise a large multigene family in the plant species. For example, in Arabidopsis, 31 expansin genes have been identified. Third, studies of expression and localization of expansin mRNA are providing new insights and hypothesis concerning the developmental roles of specific expansin genes. And fourth, sequence comparisons have led to the discovery that another group of proteins known previously as group-1 grass pollen allergens, have expansin activity. These pollen-specific proteins are closely related to a group of sequences known primarily from expressed sequence tag (EST) databases. These EST sequences, together with the group-1 pollen allergens, have now been classified as beta-expansins, whereas the original group of expansins are now classified as alpha-expansins. The alpha-expansins are described in U.S. Pat. Nos. 5,959,082 and 5,990,283 to Cosgrove et al., which are herein incorporated by reference. Beta-expansins, in general, are the subject of a previously filed U.S. patent application Ser. No. 09/071,252 filed May 1, 1998. Although these two expansin families have only about 20% amino acid identity, they are similar in size, they share a number of conserved motifs, and they have similar wall-loosening activities.
To date, most studies have focused on alpha-expansins, and limited work has been done on beta-expansins. A soybean cytokinin-induced gene known as CIM1 is now classified as a beta-expansin, but the biological function of the CIM1 protein is uncertain. The maize group-1 pollen allergen, Zea m1, has wall-loosening activity with high specificity for grass cell walls. This beta-expansin is hypothesized to aid fertilization by loosening the cell walls of the stigma and style, thereby facilitating penetration of the pollen tube. Many other beta-expansin sequences are found in the rice EST databases, and most of these sequences come from cDNA libraries made from young seedlings and other plant materials that do not contain pollen. Thus, their biological functions clearly differ from those of the group-1 pollen allergens. These so-called vegetative beta-expansins are hypothesized to function in cell enlargement and other processes where wall loosening is required. It is notable that the rice EST collection contains at least 75 entries representing at least 10 distinct beta-expansin genes. In contrast, only a single Arabidopsis EST is classified as a beta-expansin (although a total of five beta-expansin genes are found in the Arabidopsis genome). The disparity in the number of beta-expansin entries in the rice and Arabidopsis EST collection, together with the specificity of Zea m1 activity for grass walls, leads to the proposal that beta-expansins have evolved specialized function in conjunction with the evolution of the grass cell wall, which has a distinctive set of matrix polysaccharide and structural proteins compared with other land plants. If this is true, one would expect to find an abundance of beta-expansin homology in other grasses, with expression in many tissues beside pollen. In this application, we describe expansins from maize.